Cyclic peptidomimetic compounds as immunomodulators

ABSTRACT

Compounds of Formula (I) are provided: 
                         
and pharmaceutically acceptable salts and stereoisomers thereof; wherein:
 
R 1  is a side chain of amino acid Ala, Ser, Thr or Leu;
 
R 2  is a side chain of amino acid Asp, Glu, Gln or Asn;
 
[Aaa] is an amino acid residue selected from Ser, ASP, Ala, Ile, Phe, Trp, Lys, Glu or Thr;
 
R 3  is hydrogen or alkyl;
 
each of R 4  and R 4 ′ independently are hydrogen or alkyl;
 
both R a  and R a ′ are hydrogen; or together are an oxo (═O) group;
 
both R b  and R b ′ are hydrogen; or together are an oxo (═O) group;
 
L is
 
                         
X is CH 2 , O or S;
 
R 5  is hydrogen or alkyl;
 
m is an integer from 1 to 3; and
 
n is an integer from 2 to 20.
 
The compounds are useful for treatment of disorders via immunopotentiation comprising inhibition of immunosuppressive signal induced due to PD-1, PD-L1, or PD-L2 and therapies using them.

CROSS-REFERENCE TO RELATED APPLICATIONS

This nonprovisional application claims the benefit of priority under 35U.S.C. §119(a) of Indian provisional application number 4010/CHE/2013,filed on Sep. 6, 2013, which is hereby incorporated by reference.

BACKGROUND OF THE INVENTION

1. Technical Field of the Invention

The present invention relates to cyclic peptidomimetic compoundstherapeutically useful as immune modulators. The invention also relatesto pharmaceutical compositions comprising said cyclic peptidomimeticcompounds as therapeutic agents.

2. Description of the Related Art

Programmed cell death-1 (PD-1) is a member of the CD28 superfamily thatdelivers negative signals upon interaction with its two ligands, PD-L1or PD-L2. PD-1 and its ligands are broadly expressed and exert a widerrange of immunoregulatory roles in T cells activation and tolerancecompared with other CD28 members. PD-1 and its ligands are involved inattenuating infectious immunity and tumor immunity, and facilitatingchronic infection and tumor progression. The biological significance ofPD-1 and its ligand suggests the therapeutic potential of manipulationof PD-1 pathway against various human diseases (Ariel Pedoeem et al.,Curr Top Microbiol Immunol. (2011); 350:17-37).

T-cell activation and dysfunction relies on direct and modulatedreceptors. Based on their functional outcome, co-signaling molecules canbe divided as co-stimulators and co-inhibitors, which positively andnegatively control the priming, growth, differentiation and functionalmaturation of a T-cell response (Li Shi, et al., Journal of Hematology &Oncology 2013, 6:74).

Therapeutic antibodies that block the programmed cell death protein-1(PD-1) immune checkpoint pathway prevent T-cell down regulation andpromote immune responses against cancer. Several PD-1 pathway inhibitorshave shown robust activity in various phases of on-going clinical trials(RD Harvey, Clinical Pharmacology & Therapeutics (2014); 96 2, 214-223).

Programmed death-1 (PD-1) is a co-receptor that is expressedpredominantly by T cells. The binding of PD-1 to its ligands, PD-L1 orPD-L2, is vital for the physiological regulation of the immune system. Amajor functional role of the PD-1 signaling pathway is the inhibition ofself-reactive T cells, which serve to protect against autoimmunediseases. Elimination of the PD-1 pathway can therefore result in thebreakdown of immune tolerance that can ultimately lead to thedevelopment of pathogenic autoimmunity. Conversely, tumor cells can attimes co-opt the PD-1 pathway to escape from immunosurveillancemechanisms. Therefore, blockade of the PD-1 pathway has become anattractive target in cancer therapy. Current approaches include sixagents that are either PD-1 and PD-L1 targeted neutralizing antibodiesor fusion proteins. More than forty clinical trials are underway tobetter define the role of PD-1 blockade in variety of tumor types(Hyun-Tak Jin et al., Clinical Immunology (Amsterdam, Netherlands)(2014), 153 (1), 145-152).

International applications WO 01/14557, WO 02/079499, WO 2002/086083, WO03/042402, WO 2004/004771, WO 2004/056875, WO2006121168, WO2008156712,WO2010077634, WO2011066389, WO2014055897, WO2014059173, WO2014100079 andU.S. Pat. No. 8,735,553 report PD-1 or PD-L1 inhibitory antibodies orfusion proteins.

Further, International applications, WO2011161699, WO2012/168944,WO2013144704 and WO2013132317 report peptides or peptidomimeticcompounds which are capable of suppressing and/or inhibiting theprogrammed cell death 1 (PD1) signaling pathway.

Still there is a need for more potent, better and/or selective immunemodulators of PD-1 pathway. The present invention provides cyclicpeptidomimetic compounds which are capable of suppressing and/orinhibiting the programmed cell death 1 (PD1) signaling pathway.

SUMMARY OF THE INVENTION

In accordance with the present invention, cyclic peptidomimeticcompounds or a stereoisomer thereof or a pharmaceutically acceptablesalt thereof or pharmaceutical compositions thereof are provided whichsuppress and/or inhibit the programmed cell death 1 (PD1) signallingpathway.

In one aspect, the present invention provides cyclic peptidomimeticcompounds of formula (I):

or a pharmaceutically acceptable salt or a stereoisomer thereof;wherein,

R₁ is a side chain of amino acid Ala, Ser, Thr or Leu;

R₂ is a side chain of amino acid Asp, Glu, Gln or Asn;

[Aaa] is an amino acid residue selected from Ser, Asp, Ala, Ile, Phe,Trp, Lys, Glu or Thr;

R₃ is hydrogen or alkyl;

each of R₄ and R₄′ independently are hydrogen or alkyl;

both R_(a) and R_(a)′ are hydrogen; or together are an oxo (═O) group;

both R_(b) and R_(b)′ are hydrogen; or together are an oxo (═O) group;

L is

X is CH₂, O or S;

R₅ is hydrogen or alkyl;

m is an integer from 1 to 3; and

n is an integer from 2 to 20.

In a further aspect of the present invention, there is provided apharmaceutical composition comprising a compound of formula (I) or apharmaceutically acceptable salt or a stereoisomer and processes forpreparing thereof.

In yet another aspect of the present invention, there is providedmethods for suppressing and/or inhibiting the programmed cell death 1(PD1) signaling pathway in a subject by administering cyclicpeptidomimetic compounds of formula (I) or a pharmaceutically acceptablesalt or a stereoisomer thereof or pharmaceutical compositions thereof.

In yet another aspect of the present invention, there is providedmethods for inhibiting growth of tumour cells and/or metastasis in asubject by administering cyclic peptidomimetic compounds of formula (I)or a pharmaceutically acceptable salt or a stereoisomer thereof orpharmaceutical compositions thereof.

In yet another aspect of the present invention, there is providedmethods for treating an infectious disease or a bacterial, viral andfungal infections in a subject by administering cyclic peptidomimeticcompounds of formula (I) or a pharmaceutically acceptable salt or astereoisomer thereof or pharmaceutical compositions thereof.

BRIEF DESCRIPTION OF THE DRAWINGS

So that the matter in which the above-recited features, advantages andobjects of the invention, as well as others which will become clear, areattained and can be understood in detail, more particular descriptionsand certain embodiments of the invention briefly summarized above areillustrated in the appended drawings. These drawings form a part of thespecification. It is to be noted, however, that the appended drawingsillustrate preferred embodiments of the invention and therefore are notto be considered limiting in their scope.

FIGS. 1A-1C depict the chemical synthetic scheme for Compound 1.

FIGS. 2A-2B depict the chemical synthetic scheme for Compound 2.

FIGS. 3A-3C depict the chemical synthetic scheme for Compound 3.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides cyclic peptidomimetic compounds astherapeutic agents useful for treatment of disorders viaimmunopotentiation comprising inhibition of immunosuppressive signalinduced due to PD-1, PD-L1, or PD-L2 and therapies using them.

Each embodiment is provided by way of explanation of the invention, andnot by way of limitation of the invention. In fact, it will be apparentto those skilled in the art that various modification and variations canbe made in the present invention without departing from the scope orspirit of the invention. For instance, features illustrated or describedas part of one embodiment can be used on another embodiment to yield astill further embodiment. Thus it is intended that the present inventioncover such modifications and variations as come within the scope of theappended claims and their equivalents. Other objects, features, andaspects of the present invention are disclosed in, or are obvious from,the following detailed description. It is to be understood by one ofordinary skill in the art that the present discussion is a descriptionof exemplary embodiments only, and is not to be construed as limitingthe broader aspects of the present invention.

In one embodiment, the present invention relates to compounds of formula(I)

or a stereoisomer or a pharmaceutically acceptable salt thereof;wherein;

R₁ is side chain of amino acid Ala, Ser, Thr or Leu;

R₂ is a side chain of amino acid Asp, Glu, Gln or Asn;

[Aaa] is an amino acid residue Ser, Asp, Ala, Ile, Phe, Trp, Lys, Glu,or Thr;

R₃ is hydrogen or alkyl;

each of R₄ and R₄′ independently are hydrogen or alkyl;

both R_(a) and R_(a)′ are hydrogen; or together are an oxo (═O) group;

both R_(b) and R_(b)′ are hydrogen; or together are an oxo (═O) group;

L is

X is CH₂, O or S;

R₅ is hydrogen or alkyl;

m is an integer from 1 to 3; and

n is an integer from 2 to 20.

In a particular embodiment of the compounds of formula (I), theinvention comprises a particular series of compounds of formula (IA):

wherein, R₁, R₂, R₃, R₄, R₄′, R_(a), R_(a)′, R_(b), R_(b)′ and [Aaa] aresame as defined in formula (I).

In a particular embodiment of the compounds of formula (I), theinvention comprises a particular series of compounds of formula (IB):

wherein, R₁, R₂ and [Aaa] are same as defined in formula (I).

In yet another embodiment of the compounds of formula (I), the inventioncomprises a particular series of compounds of formula (IC):

wherein, R₁, R₂ and [Aaa] are same as defined in formula (I).

In yet another embodiment, the present invention provides compounds offormula (I),

wherein,

R₁ is a side chain of amino acid Ser or Thr;

R₂ is a side chain of amino acid of Asp, Asn or Glu;

[Aaa] is an amino acid residue selected from Ser or Thr;

R₃, R₄ and R₄′ independently are hydrogen;

both R_(a) and R_(a)′ together are an oxo (═O) group;

both R_(b) and R_(b)′ together are an oxo (═O) group;

L is —C(O)—(CH₂)_(m)—(X—CH₂—CH₂)_(n)—NH—;

X is CH₂ or O;

m is an integer from 1 to 3; and

n is an integer from 2 to 20;

or a pharmaceutically acceptable salt or a stereoisomer thereof.

The embodiments below are illustrative of the present invention and arenot intended to limit the claims to the specific embodimentsexemplified.

In one embodiment, specifically provided are compounds of the formula(I), in which L is

wherein X, n and R₅ are the same as defined in formula (I).

In another embodiment, specifically provided are compounds of theformula (I), in which L is

wherein m, n and R₅ are the same as defined in formula (I).

In yet another embodiment, specifically provided are the compounds ofthe formula (I), in which L is —C(O)—(CH₂)_(m)—(X—CH₂—CH₂)_(n)—NH—;wherein m, n and X are the same as defined in formula (I).

In yet another embodiment, specifically provided are compounds of theformula (I), in which L is

wherein m, X and R₅ are the same as defined in formula (I).

In yet another embodiment, specifically provided are compounds of theformula (I), in which L is —C(O)—CH₂—(OCH₂CH₂)₂—NH—.

In another embodiment, specifically provided are compounds of theformula (I), (IA), (IB) and (IC), in which R₁ is side chain of Ser.

In yet another embodiment, specifically provided are compounds of theformula (I), (IA), (IB) and (IC) in which R₁ is side chain of Thr.

In yet another embodiment, specifically provided are compounds of theformula (I), (IA) and (IB), in which R₂ is side chain of Asp.

In yet another embodiment, specifically provided are compounds of theformula (I), (IA), (IB) and (IC) in which R₂ is side chain of Asn.

In yet another embodiment, specifically provided are compounds of theformula (I), (IA), (IB) and (IC) in which R₂ is side chain of Glu.

In yet another embodiment, specifically provided are compounds of theformula (I), (IA), (IB) and (IC) in which [Aaa] is Ser.

In yet another embodiment, specifically provided are compounds of theformula (I), (IA), (IB) and (IC) in which [Aaa] is Thr.

In yet another embodiment, specifically provided are compounds of theformula (I), (IA) and (IB) in which [Aaa] is Asp.

In yet another embodiment, specifically provided are compounds of theformula (I), (IA) and (IB) in which [Aaa] is Lys or Ile.

In yet another embodiment, specifically provided are compounds of theformula (I), (IA) and (IB), in which one, more or all amino acid/sis/are D amino acid/s.

In yet another embodiment, specifically provided are compounds of theformula (I) and (IA), in which R₄′ is C₁₋₅alkyl such as methyl.

In yet another embodiment, specifically provided are compounds of theformula (I) and (IA), in which R₄ is C₁₋₅ alkyl such as methyl.

In yet another embodiment, specifically provided are compounds of theformula (I) and (IA), in which both R_(a) and R_(a)′ are hydrogen.

In yet another embodiment, specifically provided are compounds of theformula (I) and (IA), in which both R_(b) and R_(b)′ are hydrogen.

In yet another embodiment, specifically provided are compounds of theformula (I) and (IA), in which both R_(a) and R_(a)′ together representan oxo (═O) group.

In yet another embodiment, specifically provided are compounds of theformula (I) and (IA), in which both R_(b) and R_(b)′ together representan oxo (═O) group.

In yet another embodiment, specifically provided are compounds of theformula (I) and (IA), in which both R₄ and R₄′ are hydrogen.

In yet another embodiment, specifically provided are compounds of theformula (I) and (IA), in which R₃ is hydrogen.

In an embodiment, specific compounds of formula (I) without anylimitation are enumerated in Table (1):

TABLE 1 Com- pound No. Structure  1

 2

 3

 4

 5

 6

 7

 8

 9

10

11

12

13

14

or a pharmaceutically acceptable salt or a stereoisomer thereof.

In one embodiment, the present invention provides a pharmaceuticalcomposition comprising the compound as disclosed, and a pharmaceuticallyacceptable carrier or diluent.

In another embodiment, the pharmaceutical composition comprises at leastone additional pharmaceutical agent wherein the additionalpharmaceutical agent is an anticancer agent, chemotherapy agent, orantiproliferative compound.

The compounds as disclosed in the present invention are formulated forpharmaceutical administration.

In one embodiment, the present invention provides use of the compoundsas disclosed in the present invention for the preparation of amedicament for the treatment of cancer.

In one embodiment, the present invention provides use of the compoundsas disclosed in the present invention for the preparation of amedicament for the treatment of infectious diseases or bacterial, viraland fungal infections.

In one embodiment, the present invention provides a method of treatmentof cancer, wherein the method comprises administration of an effectiveamount of the compound of the present invention or of a pharmaceuticalcomposition thereof to the subject in need thereof.

In one embodiment, the present invention provides a method forinhibiting growth of tumour cells and/or metastasis by administering aneffective amount of the compound of the present invention or of apharmaceutical composition thereof to the subject in need thereof.

Representative tumour cells include cancer such as but not limited tomelanoma, renal cancer, prostate cancer, breast cancer, colon cancer andlung cancer, bone cancer, pancreatic cancer, skin cancer, cancer of thehead or neck, cutaneous or intraocular malignant melanoma, uterinecancer, ovarian cancer, rectal cancer, cancer of the anal region,stomach cancer, testicular cancer, carcinoma of the fallopian tubes,carcinoma of the endometrium, carcinoma of the cervix, carcinoma of thevagina, carcinoma of the vulva, Hodgkin's Disease, non-Hodgkin'slymphoma, cancer of the esophagus, cancer of the small intestine, cancerof the endocrine system, cancer of the thyroid gland, cancer of theparathyroid gland, cancer of the adrenal gland, sarcoma of soft tissue,cancer of the urethra, cancer of the penis, chronic or acute leukemiasincluding acute myeloid leukemia, chronic myeloid leukemia, acutelymphoblastic leukemia, chronic lymphocytic leukemia, solid tumours ofchildhood, lymphocytic lymphoma, cancer of the bladder, cancer of thekidney or ureter, carcinoma of the renal pelvis, neoplasm of the centralnervous system (CNS), primary CNS lymphoma, tumour angiogenesis, spinalaxis tumour, brain stem glioma, pituitary adenoma, Kaposi's sarcoma,epidermoid cancer, squamous cell cancer, T-cell lymphoma,environmentally induced cancers including those induced by asbestos, andcombinations of said cancers.

Still yet another embodiment of the present invention provides a methodof treatment of infection via immunopotentiation caused by inhibition ofimmunosuppressive signal induced by PD-1, PD-L1, or PD-L2, wherein themethod comprises administration of an effective amount of the compoundof the present invention or of a pharmaceutical composition thereof tothe subject in need thereof.

The infectious disease includes but not limited to HIV, Influenza,Herpes, Giardia, Malaria, Leishmania, the pathogenic infection by thevirus Hepatitis (A, B, & C), herpes virus (e.g., VZV, HSV-I, HAV-6,HSV-II, and CMV, Epstein Barr virus), adenovirus, influenza virus,flaviviruses, echovirus, rhinovirus, coxsackie virus, cornovirus,respiratory syncytial virus, mumps virus, rotavirus, measles virus,rubella virus, parvovirus, vaccinia virus, HTLV virus, dengue virus,papillomavirus, molluscum virus, poliovirus, rabies virus, JC virus andarboviral encephalitis virus, pathogenic infection by the bacteriachlamydia, rickettsial bacteria, mycobacteria, staphylococci,streptococci, pneumonococci, meningococci and conococci, klebsiella,proteus, serratia, pseudomonas, E. coli, legionella, diphtheria,salmonella, bacilli, cholera, tetanus, botulism, anthrax, plague,leptospirosis, and Lyme's disease bacteria, pathogenic infection by thefungi Candida (albicans, krusei, glabrata, tropicalis, etc.),Cryptococcus neoformans, Aspergillus (fumigatus, niger, etc.), GenusMucorales (mucor, absidia, rhizophus), Sporothrix schenkii, Blastomycesdermatitidis, Paracoccidioides brasiliensis, Coccidioides immitis andHistoplasma capsulatum, and pathogenic infection by the parasitesEntamoeba histolytica, Balantidium coli, Naegleriafowleri, Acanthamoebasp., Giardia lambia, Cryptosporidium sp., Pneumocystis carinii,Plasmodium vivax, Babesia microti, Trypanosoma brucei, Trypanosomacruzi, Leishmania donovani, Toxoplasma gondi, Nippostrongylusbrasiliensis.

The compounds of the present invention may be used as single drugs or asa pharmaceutical composition in which the compound is mixed with variouspharmacologically acceptable materials.

The pharmaceutical composition is usually administered by oral orinhalation routes, but can be administered by parenteral administrationroute. In the practice of this invention, compositions can beadministered, for example, by orally, intravenous infusion, topically,intraperitoneally, intravesically or intrathecally. Examples of theparenteral administration includes but not limited to intraarticular (inthe joints), intravenous, intramuscular, intradermal, intraperitoneal,and subcutaneous routes, include aqueous and non-aqueous, isotonicsterile injection solutions, which can contain antioxidants, buffers,bacteriostats, and solutes that render the formulation isotonic with theblood of the intended recipient, and aqueous and non-aqueous sterilesuspensions that can include suspending agents, solubilizers, thickeningagents, stabilizers, and preservatives. Oral administration, parenteraladministration, subcutaneous administration and intravenousadministration are the preferred methods of administration.

The dosage of the compounds of the present invention varies depending onage, weight, symptom, therapeutic efficacy, dosing regimen and/ortreatment time. Generally, they may be administered by oral orinhalation routes, in an amount of 1 mg to 100 mg per time, from once acouple of days, once 3 days, once 2 days, once a day to a couple oftimes a day, in the case of an adult, or continuously administered byoral or inhalation routes from 1 to 24 hours a day. Since the dosage isaffected by various conditions, an amount less than the above dosage maysometimes work well enough, or higher dosage may be required in somecases.

The compounds of the present invention may be administered incombination with other drugs for (1) complementation and/or enhancementof prevention and/or therapeutic efficacy of the preventive and/ortherapeutic drug of the present invention, (2) dynamics, absorptionimprovement, dosage reduction of the preventive and/or therapeutic drugof the present invention, and/or (3) reduction of the side effects ofthe preventive and/or therapeutic drug of the present invention.

A concomitant medicine comprising the compounds of the present inventionand other drug may be administered as a combination preparation in whichboth components are contained in a single formulation, or administeredas separate formulations. The administration by separate formulationsincludes simultaneous administration and administration with some timeintervals. In the case of the administration with some time intervals,the compound of the present invention can be administered first,followed by another drug or another drug can be administered first,followed by the compound of the present invention. The administrationmethod of the respective drugs may be the same or different.

The dosage of the other drug can be properly selected, based on a dosagethat has been clinically used. The compounding ratio of the compound ofthe present invention and the other drug can be properly selectedaccording to age and weight of a subject to be administered,administration method, administration time, disorder to be treated,symptom and combination thereof. For example, the other drug may be usedin an amount of 0.01 to 100 parts by mass, based on 1 part by mass ofthe compound of the present invention. The other drug may be acombination of two or more kind of arbitrary drugs in a properproportion. The other drug that complements and/or enhances thepreventive and/or therapeutic efficacy of the compound of the presentinvention includes not only those that have already been discovered, butthose that will be discovered in future, based on the above mechanism.

Diseases on which this concomitant use exerts a preventive and/ortherapeutic effect are not particularly limited. The concomitantmedicine can be used for any diseases, as long as it complements and/orenhances the preventive and/or therapeutic efficacy of the compound ofthe present invention.

The compound of the present invention can be used with an existingchemotherapeutic concomitantly or in a mixture form. Examples of thechemotherapeutic include an alkylation agent, nitrosourea agent,antimetabolite, anticancer antibiotics, vegetable-origin alkaloid,topoisomerase inhibitor, hormone drug, hormone antagonist, aromataseinhibitor, P-glycoprotein inhibitor, platinum complex derivative, otherimmunotherapeutic drugs and other anticancer drugs. Further, it can beused with a cancer treatment adjunct, such as a leucopenia (neutropenia)treatment drug, thrombocytopenia treatment drug, antiemetic and cancerpain intervention drug, concomitantly or in a mixture form.

In one embodiment, the compound(s) of the present invention can be usedwith other immunomodulators and/or a potentiating agent concomitantly orin a mixture form. Examples of the immunomodulator include variouscytokines, vaccines and adjuvants. Examples of these cytokines, vaccinesand adjuvants that stimulates immune responses include but not limitedto GM-CSF, M-CSF, G-CSF, interferon-α, β, or γ, IL-1, IL-2, IL-3, IL-12,Poly (I:C) and C_(p)G.

In another embodiment, the potentiating agents includes cyclophosphamideand analogs of cyclophosphamide, anti-TGFβ and Imatinib (Gleevac), amitosis inhibitor, such as paclitaxel, Sunitinib (Sutent) or otherantiangiogenic agents, an aromatase inhibitor, such as letrozole, an A2aadenosine receptor (A2AR) antagonist, an angiogenesis inhibitor,anthracyclines, oxaliplatin, doxorubicin, TLR4 antagonists, and IL-18antagonists.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as is commonly understood by one of skill in artto which the subject matter herein belongs. As used herein, thefollowing definitions are supplied in order to facilitate theunderstanding of the present invention.

As used herein the term “alkyl” refers to a hydrocarbon chain radicalthat includes solely carbon and hydrogen atoms in the backbone,containing no unsaturation, having from one to twenty carbon atoms(i.e., C₁₋₂₀ alkyl) or one to ten carbon atoms (i.e., C₁₋₁₀ alkyl) orone to five carbon atoms (i.e., C₁₋₅ alkyl) and which is attached to therest of the molecule by a single bond, e.g., including but not limitedto methyl, ethyl, propyl, butyl, isobutyl, sec-butyl, tert-butyl,isopentyl or neopentyl. Unless set forth or recited to the contrary, allalkyl groups described or claimed herein may be straight chain orbranched, substituted or unsubstituted.

As used herein, the term “amino acid” refers to amino acids having L orD stereochemistry at the alpha carbon.

As used herein, the term ‘compound(s)’ refers to the compounds disclosedin the present invention.

As used herein, the term “comprise” or “comprising” is generally used inthe sense of include, that is to say permitting the presence of one ormore features or components.

As used herein, the term “including” as well as other forms, such as“include”, “includes,” and “included,” is not limiting.

“Pharmaceutically acceptable salt” is taken to mean an activeingredient, which comprises a compound of the formula (I) in the form ofone of its salts, in particular if this salt form imparts improvedpharmacokinetic properties on the active ingredient compared with thefree form of the active ingredient or any other salt form of the activeingredient used earlier. The pharmaceutically acceptable salt form ofthe active ingredient can also provide this active ingredient for thefirst time with a desired pharmacokinetic property which it did not haveearlier and can even have a positive influence on the pharmacodynamicsof this active ingredient with respect to its therapeutic efficacy inthe body.

“Pharmaceutically acceptable” means that which is useful in preparing apharmaceutical composition that is generally safe, non-toxic, andneither biologically nor otherwise undesirable and includes that whichis acceptable for veterinary as well as human pharmaceutical use.

The term “stereoisomer” refers to any enantiomers, diastereomers, orgeometrical isomers of the compounds of formula (I), wherever they arechiral or when they bear one or more double bond. When the compounds ofthe formula (I) and related formulae are chiral, they can exist inracemic or in optically active form. Since the pharmaceutical activityof the racemates or stereoisomers of the compounds according to theinvention may differ, it may be desirable to use the enantiomers. Inthese cases, the end product or even the intermediates can be separatedinto enantiomeric compounds by chemical or physical measures known tothe person skilled in the art or even employed as such in the synthesis.In the case of racemic amines, diastereomers are formed from the mixtureby reaction with an optically active resolving agent. Examples ofsuitable resolving agents are optically active acids such as the R and Sforms of tartaric acid, diacetyltartaric acid, dibenzoyltartaric acid,mandelic acid, malic acid, lactic acid, suitable N-protected amino acids(for example N-benzoylproline or N-benzenesulfonylproline), or thevarious optically active camphorsulfonic acids. Also advantageous ischromatographic enantiomer resolution with the aid of an opticallyactive resolving agent (for example dinitrobenzoylphenylglycine,cellulose triacetate or other derivatives of carbohydrates or chirallyderivatised methacrylate polymers immobilised on silica gel).

The term “subject” includes mammals (especially humans) and otheranimals, such as domestic animals (e.g., household pets including catsand dogs) and non-domestic animals (such as wildlife).

“Therapeutically effective amount” or “efficient amount” refers tosufficient amount of the compound(s) of the present invention that (i)treats or prevents the particular disease, disorder or syndrome (ii)attenuates, ameliorates or eliminates one or more symptoms of theparticular disease, disorder or syndrome or (iii) prevents or delays theonset of one or more symptoms of the particular disease, disorder orsyndrome described herein. In the case of cancer, the therapeuticallyeffective amount of the drug may decrease the number of cancer cells;decrease the cancer size; inhibit (i.e., slow to some extent andalternatively stop) cancer cell infiltration into peripheral organs;suppress (i.e., slow to some extent and alternatively stop) tumormetastasis; inhibit, to some extent, tumor growth; and/or relieve tosome extent one or more of the symptoms associated with the cancer. Inthe case of infectious disease states, the therapeutic effective amountis an amount sufficient to decrease or alleviate an infectious diseases,the symptoms of an infections caused by bacterial, viral and fungal.

Naturally-occurring amino acids are identified throughout thespecification by the conventional three-letter abbreviations indicatedin the below table 2:

TABLE 2 Name 3-letter code Alanine Ala Asparagine Asn Aspartic acid AspGlutamic acid Glu Glutamine Gln Isoleucine Ile Leucine Leu Lysine LysPhenylalanine Phe Serine Ser Threonine Thr Tryptophan Trp

The abbreviations used in the entire specification may be summarizedherein below with their particular meaning.

° C. (degree Celsius); δ (delta); % (percentage); brine (NaCl solution);bs or brs (Broad singlet); Bzl (Benzyl); Cbz (Carboxybenzyl); Cbz-Cl(Benzyl chloroformate); CH₂Cl₂/DCM (Dichloromethane); Cs₂CO₃ (Cesiumcarbonate); DMF (Dimethyl formamide); DMSO (Dimethyl sulfoxide);DIPEA/DIEA (N,N-Diisopropyl ethylamine); DMSO-d₆ (Deuterated DMSO); d(Doublet); EtOAc (Ethyl acetate); Et₂NH (Diethylamine); Fmoc(Fluorenylmethyloxycarbonyl); Fmoc-Cl (Fluorenylmethyloxycarbonylchloride) g or gr (gram); H or H₂ (Hydrogen); H₂O (Water); HOBt/HOBT(1-Hydroxy benzotriazole); HCl (Hydrochloric acid); h or hr (Hours);HATU (2-(1H-7-Azabenzotriazol-1-yl)-1,1,3,3-tetramethyl uraniumhexafluoro phosphate methanaminium); Hz (Hertz); HPLC (High-performanceliquid chromatography); LCMS (Liquid chromatography mass spectroscopy);MeOH/CH₃OH (Methanol); mmol (Millimoles); M (Molar); μl/μL (Microliter);mL (Milliliter); mg (Milligram); min (minutes); m (Multiplet); mm(Millimeter); MHz (Megahertz); MS (ES) (Mass spectroscopy-electrospray); min (Minutes); Na (Sodium); NaOBu^(t) (Sodium tert-butoxide);NH₂NH₂.H₂O (Hydrazine hydrate); Na₂SO₄ (Sodium sulphate); N₂ (Nitrogen);NMR (Nuclear magnetic resonance spectroscopy); NaHCO₃ (Sodiumbicarbonate); Pd—C (Palladiun on carbon); 10% Pd/C (10% palladiumactivated carbon); Pd(OH)₂ (palladium hydroxide); PD-L1 (Programmeddeath-ligand 1); PD-L2 (Programmed cell death 1 ligand 2); prepHPLC/prep-HPLC (Preparative High-performance liquid chromatography);PyBOP (Benzotriazol-1-yloxy)tripyrrolidinophosphoniumhexafluorophosphate); RT/rt (room temperature); S (Singlet); ^(t)Bu/tBu(Tertiary butyl) TEA/Et₃N (Triethyl amine); TFA (Trifluoroaceticacid);TLC (Thin Layer Chromatography); THF (Tetrahydrofuran); TFA/CF₃COOH(Trifluoro acetic acid); t (Triplet); t_(R)=(Retention time), etc.

An embodiment of the present invention provides the preparation ofcompounds of formula (I) according to the procedures of the followingexamples, using appropriate materials. Those skilled in the art willunderstand that known variations of the conditions and processes of thefollowing preparative procedures can be used to prepare these compounds.Moreover, by utilizing the procedures described in detail, one ofordinary skill in the art can prepare additional compounds of thepresent invention.

The starting materials are generally available from commercial sourcessuch as Sigma-Aldrich, USA or Germany; Chem-Impex USA; G.L. Biochem,China and Spectrochem, India.

Purification and Characterization of Compounds

Analytical HPLC method: Analytical HPLC was performed using on ZIC HILIC200 A° column (4.6 mm×250 mm, 5 μm), Flow rate: 1.0 mL/min. The elutionconditions used are: Buffer A: 5 mmol ammonium acetate, Buffer B:Acetonitrile, Equilibration of the column with 90% buffer B and elutionby a gradient of 90% to 40% buffer B during 30 min.

Preparative HPLC Method A: The crude material was purified bypreparative HPLC using ZIC HILIC 200 A° column (21.2 mm×150 mm, 5 μm).The elution conditions used are Eluent: A: 5 mmol ammonium acetate B:Acetonitrile, Flow rate: 18 mL/min. The compound was eluted by gradientelution 0-3 min=90% buffer B, 3-20 min=90-40% buffer B with a flow rateof 20 mL/min.

Preparative HPLC Method B: Prep HPLC was performed using on ZIC HILIC200 A° column (10 mm×250 mm, 5 μm), Flow rate: 5.0 mL/min. The elutionconditions used are: Buffer A: 5 mmol ammonium acetate, Buffer B:Acetonitrile, Equilibration of the column with 90% buffer B and elutionby a gradient of 90% to 40% buffer B during 20 min. LCMS was performedon AP1 2000 LC/MS/MS triple quad (Applied biosystems) with Agilent 1100series HPLC with G1315 B DAD, using Mercury MS column or using AgilentLC/MSD VL single quad with Agilent 1100 series HPLC with G1315 B DAD,using Mercury MS column or using Shimadzu LCMS 2020 single quad withProminence UFLC system with SPD-20 A DAD.

EXAMPLE 1 Synthesis of Compound 1

FIGS. 1A-1C illustrate Steps 1a to 1c.

Step 1a: Sodium hydroxide (12.2 g, 305 mmol) and Cbz-Cl (12.5 g, 73mmol) were added to a solution of compound 1a (10.0 g, 61 mmol) in water(100 mL) and stirred at room temperature for 3 h. The completeness ofthe reaction was confirmed by TLC analysis. The reaction mass waspartitioned between citric acid solution and ethyl acetate. Organiclayer was washed with water, brine, dried over Na₂SO₄ and evaporatedunder reduced pressure to yield 11 g of compound 1b (Yield: 61.1%).LCMS: 298.0 (M+H)⁺.

Step 1b: DIPEA (3.5 g, 26.8 mmol) was added slowly to a stirred solutionof compound 1b (4.0 g, 13.4 mmol) and HATU (5.6 g, 14.7 mmol) in DMF (50mL) and was allowed to stir at room temperature for 5 more min. To theabove reaction mixture L-Ser(^(t)Bu)-OMe.HCl (3.5 g, 20.1 mmol) wasadded slowly and stirred at room temperature for 12 h. The completenessof the reaction was confirmed by TLC analysis. The reaction mixture wasquenched with ice, precipitated solid was filtered and re-crystallizedwith CH₂Cl₂ to yield 6 g of compound 1c, LCMS: 454.8 (M+H)⁺.

Step 1c: 99% Hydrazine hydrate solution (10 mL) was added slowly to astirred solution of compound 1c (6 g) in methanol (50 mL) and stirred atroom temperature for 2 h. The completion of the reaction was confirmedby TLC. The reaction mixture on evaporation under reduced pressureyielded 5.8 g of compound 1d. LCMS: 455.0 (M+H)⁺.

Step 1d: DIPEA (3.3 g, 25.4 mmol) was added slowly to a stirred solutionof compound 1d (5.8 g, 12.7 mmol), HATU (5.8 g, 15.2 mmol) in DMF (50mL) and was allowed to stir at room temperature for 5 min.Fmoc-L-Asn-OH(4.9 g, 14.0 mmol) was further added to reaction mixtureand stirred at room temperature for 12 h. The completeness of thereaction was confirmed by TLC analysis. The reaction mixture was thenquenched with ice, precipitated solid was filtered and re-crystallizedwith CH₂Cl₂ to yield 5.9 g of compound 1e, LCMS: 791.0 (M+H)⁺.

Step 1e: Fmoc group of compound 1e [(5.9 g in CH₂Cl₂ (60 mL)] wasdeprotected using diethylamine (60 mL) and the completion of thereaction was confirmed by TLC analysis. The reaction mixture onevaporation under reduced pressure yielded 1.6 g of compound 1f. LCMS:568.8 (M+H)⁺.

Step 1f: Compound 11 (1.3 g, 3.0 mmol) and compound 1f (1.60 g, 2.8mmol) was dissolved in THF (10 mL) and stirred at room temperature.Coupling was initiated by the addition of triethylamine (0.57 g, 5.6mmol) to the above reaction mixture and the reaction was allowed to stirfor 12 h at room temperature. The completeness of the reaction wasconfirmed by TLC analysis. Organic layer was washed with NaHCO₃, citricacid solution, brine, dried over Na₂SO₄ and evaporated under reducedpressure to yield 0.45 g of compound 1g.

Step 1g: Cbz group and benzyl ester deprotection was carried out oncompound 1g (0.45 g) in methanol using palladium hydroxide (0.5 g) for 1h at room temperature. The completeness of the reaction was confirmed byTLC analysis. Palladium hydroxide was removed by Celite® bed filtrationand the filtrate was evaporated under reduced pressure to yield 0.34 gof compound 1h. LCMS: 636.0 (M+H)⁺.

Step 1h: Cyclization of compound 1h (0.1 g, 0.15 mmol) was carried outusing HOBT (0.06 g, 0.47 mmol) and PyBOP (0.24 g, 0.47 mmol) in THF (50mL). The reaction was initiated by slow addition of DIPEA (0.06 g, 0.47mmol) and further stirred at room temperature for 12 h. The reactionmixture was evaporated and washed with diethyl ether to yield 0.05 g ofcompound 1i. LCMS: 617.9 (M+H)⁺.

Step 1i: The acid sensitive protecting group on compound 1i [(0.08 g) inCH₂Cl₂ (0.9 mL)] was removed using TFA (0.9 mL). The reaction mixturewas stirred at room temperature for 4 h, followed by evaporation andwashing with diethyl ether yielded 0.05 g of crude compound 1. The crudesolid material was purified using preparative HPLC method-A describedunder experimental conditions (yield: 9 mg). LCMS: 506.4 (M+H)⁺; HPLC:t_(R)=12.3 min.

Synthesis of compound 1l (NO₂—C₆H₄—OCO-Thr(^(t)Bu)-OBzl): To a solutionof Fmoc-Thr(^(t)Bu)-OH (15.0 g, 37.7 mmol) in 100.0 mL of DMF, Cs₂CO₃(14.8 g, 45.2 mmol) was added and the resulting mixture was cooled to 0°C. To the cooled reaction mixture benzyl bromide (7.74 g, 345.2 mmol)was added and the solution was stirred for 30 min at ice coldtemperature followed by room temperature for 12 h. The reaction mixturewas further concentrated under reduced pressure and diluted with ethylacetate (150 mL). The organic layer was washed with water (2×100 mL),brine (1×100 mL) and dried over Na₂SO₄. The filtered solution wasconcentrated and purified by silica gel column chromatography (Eluent:0-30% ethyl acetate in Hexane) to yield 18.5 g of intermediate 1j as awhite solid. LCMS: 433.1 (M-O^(t)Bu+H)⁺. Fmoc group on compound 1j (10.0g, 20.5 mmol) in CH₂Cl₂ (40.0 mL) was deprotected by stirring it withdiethylamine (40.0 mL) for 1 h at room temperature. The resultingsolution was concentrated in vacuum and the thick-residue was purifiedby column chromatography over neutral alumina (Eluent: 0-50% ethylacetate in hexane then 0-5% methanol in chloroform) to yield 3.5 g ofintermediate 1k (Yield: 70%). LCMS: 266.5 (M+H)⁺. TEA (1.2 g, 12.0 mmol)was added to a stirred solution of intermediate 1k (1.6 g, 6.0 mmol) inCH₂Cl₂ (30 mL). Solution of 4-nitrophenyl chloroformate (1.3 g, 6.6mmol) in CH₂Cl₂ (10 mL) was added to the above stirred solution and thereaction was continued for 12 h at room temperature. The completion ofthe reaction was confirmed by TLC analysis After completion of reaction,the reaction mixture was diluted with CH₂Cl₂ (50 mL) and washed with 1.0M of sodium bi sulphate (50 mL×2) and 1.0 M sodium carbonate (50 mL×2),dried over Na₂SO₄ and evaporated under reduced pressure to yield crudecompound 1l, which was further purified by silica gel columnchromatography (eluent: 0-20% ethyl acetate in hexane) to yield 0.8 g ofcompound 1l. ¹H NMR (DMSO-d₆, 300 MHz): δ 1.04 (s, 9H), 1.16 (d, 3H),4.11 (m, 1H), 5.11 (m, 3H), 6.91 (d, 2H), 7.40 (m, 5H), 8.10 (d, 2H),8.26 (brs, 1H).

EXAMPLE 2 Synthesis of Compound 2

FIGS. 2A-2B illustrates Steps 2a and 2h.

Step 2a: DIPEA (2.71 g, 21 mmol) was added slowly to a stirred solutionof compound 1b (3.12 g, 10.5 mmol), HATU (4.41 g, 11.6 mmol) in DMF (30mL) and was allowed to stir at room temperature for 5 min. To the abovereaction mixture D-Thr(^(t)Bu)-OMe.HCl (2.0 g, 10.5 mmol) was addedslowly and stirred at room temperature for 12 h. The completeness of thereaction was confirmed by TLC analysis. The reaction mixture was thenquenched with ice, precipitate was filtered and re-crystallized withCH₂Cl₂ to yield 4.2 g of compound 2a. LCMS: 491.2 (M+Na)⁺.

Step 2b: 99% Hydrazine hydrate solution (5 mL) was added slowly to astirred solution of compound 2a (4.2 g) in methanol (40 mL) and thecompletion of the reaction was confirmed by TLC analysis. The reactionmixture on evaporation under reduced pressure yielded 4.2 g of compound2b (Yield: 90%). LCMS: 469.4 (M+H)⁺.

Step 2c: DIPEA (2.7 mL, 20.9 mmol) was added slowly to a stirredsolution of compound 2b (4.9 g, 10.5 mmol), HATU (4.8 g, 12.5 mmol) inDMF (50 mL). To the above stirred solution, Fmoc-D-Asn(Trt)-OH (6.2 g,10.5 mmol) was added and further stirred at room temperature for 12h.The completeness of the reaction was confirmed by TLC analysis. Thereaction mixture was diluted with EtOAc (30 mL) and washed with 1.0 Msodium carbonate (20 mL×2), 10% citric acid (20 mL×2), water (20 mL×2),dried over Na₂SO₄ and evaporated under reduced pressure to yield 10 g ofcrude intermediate 2c and was further purified by silica gel columnchromatography (eluent: 0-5% MeOH in EtOAc) to yield 5 g of compound 2c.LCMS: 1047.7 (M+H)⁺.

Step 2d: Fmoc deprotection of compound 2c [(3.2 g) in CH₂Cl₂ (10 mL)]was carried out using diethylamine (10 mL). The completion of thereaction was confirmed by TLC analysis. The resulting solution onevaporation under reduced pressure yielded 1.2 g of compound 2d.

Step 2e: Triethylamine (0.32 g, 3.2 mmol) was added slowly to initiatethe coupling of compound 2d (1.3 g, 1.6 mmol) and compound 2h (0.79 g,1.9 mmol) in THF (20 mL). The resulting solution was further allowed tostir for 12 h at room temperature and completeness of the reaction wasconfirmed by TLC analysis. Organic layer was washed with NaHCO₃, citricacid solution, brine, dried over Na₂SO₄ and evaporated under reducedpressure to yield 1.3 g of compound 2e.

Step 2f: Cbz group and benzyl ester deprotection was carried out oncompound 2e (1.3 g) in methanol using palladium hydroxide (1.0 g) for 1h at room temperature. The completeness of the reaction was confirmed byTLC analysis. Palladium hydroxide was removed by Celite® bed filtrationand the filtrate was evaporated under reduced pressure to yield 0.45 gof compound 2f. LCMS: 878.4 (M+H)⁺.

Step 2g: DIPEA (0.2 g, 1.5 mmol) was added slowly to a stirred solutionof compound 2f (0.45 g, 0.51 mmol), HOBT (0.21 g, 1.53 mmol) and PyBOP(0.8 g, 1.53 mmol) in THF (200 mL). The reaction mixture was furtherstirred at room temperature for 12 h. The completeness of the reactionwas confirmed by TLC analysis. The reaction mixture was evaporated andwashed with diethyl ether to yield 0.41 g of intermediate 2g. LCMS:860.7 (M+H)⁺.

Step 2h: To a solution of compound 2g (0.4 g) in CH₂Cl₂ (5 mL),trifluoroacetic acid (5 mL) and catalytic amount of triisopropylsilanewere added and stirred for 3 h at room temperature. The resultingsolution was concentrated in vacuum to yield 0.2 g of crude compound 2.The crude solid material was purified (yield: 10 mg) using preparativeHPLC method-B described under experimental conditions. LCMS: 506.6(M+H)⁺; HPLC: t_(R)=12.4 min.

Synthesis of compound 2h (NO₂—C₆H₄—OCO-D-Ser(^(t)Bu)-OBzl)

The compound was synthesised using similar procedure as exemplified in(example 1, compound 1l) using Fmoc-D-Ser(^(t)Bu)-OH instead ofFmoc-Thr(^(t)Bu)-OH to yield 1g crude material of 2h.

EXAMPLE 3 Synthesis of Compound 3

FIGS. 3A-3C illustrates Steps 3a and 3k.

Step 3a: To a stirred solution of compound 1a (5.0 g, 30.6 mmol) in1,4-Dioxane (50 mL), Sodium carbonate (8.12 g, 76.5 mmol, dissolved in10 mL water) and (Boc)₂O (9.98 mL, 45.7 mmol) were added and stirred atroom temperature for 12 h. The progress of reaction was monitored byTLC. The reaction mass was partitioned between diethyl ether and water.Then aqueous layer was made acidic (pH=3) by 3N HCl solution and wasextracted with DCM (2×200 mL). Organic layer was washed with water,brine, dried over Na₂SO₄ and evaporated under reduced pressure to yield50 g of pure 3a (Yield: 62.1%). LCMS: 263.0 (M+H)⁺.

Step 3b: DIPEA (6.5 mL, 37.8 mmol) was added slowly to a stirredsolution of compound 3b (5 g, 12.6 mmol), compound 3c (2.72 g, 15 mmol),HOBt (2.55 g, 18.9 mmol) and EDC.HCl (3.62 g, 18.9 mmol) in DMF (75 mL)at 0° C. The reaction mixture was further stirred at room temperaturefor 12 h. The progress of the reaction was confirmed by TLC analysis.The reaction mass was partitioned between ethyl acetate and water.Organic layer was washed with water, brine, dried over Na₂SO₄ andevaporated under reduced pressure to yield crude. The crude compound waspurified by silica gel (60-120 mesh) column chromatography usinghexane/ethyl acetate (40:60) as elute to yield 5.2 g of compound 3d,(Yield: 71.0%). LCMS: 560.6 (M+H)⁺.

Step 3c: To a stirred solution of compound 3d (5 g, 8.9 mmol) in dryDCM, diethylamine (50 mL) was added dropwise at −10° C. and stirred for1 h at room temperature. After completion of reaction, the mixture wasevaporated under reduced pressure to give crude compound. The crude waspurified with (1:1) n-pentane/diethyl ether wash and dried under highvacuum to yield 3.5 g of compound 3e. LCMS: 338.58 (M+H)⁺.

Step 3d: NMM (1.4 mL, 14.0 mmol) was added slowly to a stirred solutionof compound 3a (3 g, 11.3 mmol), compound 3e (4.3 g, 12.9 mmol) and HATU(6.5 g, 17.1 mmol) in DMF (75 mL) at 0° C. The reaction mixture wasfurther stirred for 6 h at room temperature. Progress of reaction wasmonitored by TLC. After completion, the reaction mass was partitionedbetween ethyl acetate and water. Organic layer was washed with water,brine, dried over Na₂SO₄ and evaporated under reduced pressure to givecrude compound. The crude was purified by silica gel columnchromatography (Eluent: 50% hexane in ethyl acetate) to yield 4.8 g ofcompound 3f, LCMS: 583.7 (M+H)⁺.

Step 3e: To a stirred solution of compound 3f (4.5 g, 7.7 mmol) in MeOH,Pd/C (2.0 g) was added slowly and stirred under H₂ atmosphere for 4 h atroom temperature. Progress of reaction was monitored by TLC. Aftercompletion, the reaction mass was filtered through Celite® and washedwith MeOH (2×150 mL). The resulting filtrate was evaporated underreduced pressure to yield 3 g of compound 3g LCMS: 448.6 (M+H)⁺.

Step 3f: To a stirred solution of Cbz-D-Asn-OH (1.78 g, 6.7 mmol) andcompound 3g (3.0 g, 6.8 mmol) in DMF (75 mL), DCC (4.12 g, 20.3 mmol)and HOBT (1.8 g, 13.5 mmol) were added slowly at 0° C. The reactionmixture was stirred for 48 h at room temperature. Progress of reactionwas monitored by TLC. After completion, the reaction mass waspartitioned between ethyl acetate and water. Organic layer was washedwith water, brine, dried over Na₂SO₄ and evaporated under reducedpressure to yield crude. The crude compound was purified by silica gel(60-120 mesh) column chromatography (Eluent: 4% MeOH in DCM) to yield2.5 g of Compound 3h, LCMS: 697.50 (M+H)⁺.

Step 3g: To a stirred solution of compound 3h (2.5 g, 3.6 mmol) in MeOH(50 mL), Pd/C (1.2 g) was added and stirred under H₂ atmosphere for 4 hat room temperature. Progress of reaction was monitored by TLC. Aftercompletion, the reaction mass was filtered through Celite® and washedwith MeOH (2×150 mL). The resulting filtrate was evaporated underreduced pressure to yield 1.5 g of compound 3i, LCMS: 563.6 (M+H)⁺.

Step 3h: Compound 3i (1.3 g, 2.3 mmol), TEA (0.43 mL, 3.5 mmol) in DMF(25 mL) and was added dropwise slowly to a solution of 3j (1.1 g, 2.6mmol) at −10° C. The mixture was further stirred at room temperature for2 h. Progress of reaction was monitored by TLC. After completion, thereaction mass was partitioned between ethyl acetate and water. Organiclayer was washed with NaHCO₃, citric acid solution, brine, then organiclayer was dried over Na₂SO₄ and evaporated under reduced pressure togive crude. The crude was purified by silica gel (60-120 mesh) columnchromatography (hexane/ethyl acetate (10:90) as elute) to yield 1.2 g ofcompound 3k. LCMS: 840.6 (M+H)⁺.

Step 3i: To a solution of compound 3k (1.0 g, 1.2 mmol) in CH₂Cl₂ (10mL), trifluoroacetic acid (10 mL) and catalytic amount oftriisopropylsilane were added and stirred for 3 h at room temperature toremove the acid sensitive protecting groups. The resulting solution wasconcentrated in vacuum and washed with diethyl ether to afford 1.0 g ofcrude compound 3l, LCMS: 628.65 (M+H)⁺.

Step 3j: To a stirred solution of compound 3l (1.0 g, 1.5 mmol) in THF,PyBOP (2.4 g, 4.7 mmol), HOBT (0.6, 4.7 mmol) and DIPEA (0.8 mL, 4.7mmol) were added slowly and stirred for 12 h at room temperature. Thereaction mass was partitioned between water and ethyl acetate. Organiclayer was washed with water, brine, dried over Na₂SO₄ and evaporatedunder reduced pressure to give crude. Crude compound was washed withdiethyl ether to yield 0.8 g of compound 3m, LCMS: 610.5 (M+H)⁺.

Step 3k: To a stirred solution of compound 3m (0.8 g, 1.3 mmol) in MeOH(30 mL), Pd(OH)₂ (0.4 g) was added and stirred under H₂ atmosphere for 4h at room temperature. Progress of reaction was monitored by TLC. Aftercompletion, the reaction mass was filtered through Celite® and washedwith MeOH (2×150 mL). The resulting filtrate was evaporated underreduced pressure to yield 0.7 g of compound 3. LCMS: 520.5 (M+H)⁺; HPLC:t_(R)=9.9 min.

Synthesis of Intermediate 3c: To a stirred solution of compound 3n (4 g,88.3 mmol) and compound 3o (10.2 mL, 71.2 mmol) in DCM (150 mL), TEA(14.4 mL, 105 mmol) was added dropwise at −78° C. The reaction mixturewas allowed to attain room temperature and stirred for 12h. Progress ofreaction was monitored by TLC. After completion, the reaction mass waspartitioned between water and DCM. Organic layer was washed with water,brine, dried over Na₂SO₄ and evaporated under reduced pressure to yieldcrude compound and was purified by silica gel (60-120 mesh) columnchromatography (Eluent: 80% ethyl acetate in hexane) to yield 10 g ofCompound 3c, LCMS: 181.18 (M+H).

Synthesis of 3j (NO₂—C₆H₄—OCO-D-Ser(Bzl)-O^(t)Bu): The compound wassynthesised using similar procedure as exemplified in (example 1,compound 1k) using Fmoc-D-Ser(Bzl)-O^(t)Bu instead ofFmoc-Thr(^(t)Bu)-OH to yield 1.5 g of compound 3j.

EXAMPLE 4 Synthesis of Compound 4

The compound was synthesised using similar procedure as depicted inExample 2 (compound 2) using 7-aminoheptanoic acid instead compound 1ato yield 0.3 g crude material of the title compound. The crude solidmaterial was purified using preparative HPLC described underexperimental conditions. LCMS: 488.2 (M+H)⁺; HPLC: t_(R)=11.9 min.

The compounds in Table 3 below were prepared based on the syntheticprocedures described above.

TABLE 3 Compound LCMS HPLC No. Structure (M + H)⁺ (t_(R) in min.)  5

518.2 9.7  6

534.2 12.4   7

532.9 —  8

520.2  9.03  9

492.1 12.9  10

520.2 11.23 11

507.2 11.96 12

493.2  8.35 13

521.3 12.2  14

535.4 11.2 

The compounds shown in below table 4, which can be prepared by followingsimilar procedure as described above with suitable modification known tothe one ordinary skilled in the art are also included in the scope ofthe present application.

TABLE 4 Compound No. Structure 15

16

17

18

19

20

21

22

23

24

25

and

EXAMPLE 5 Rescue of Mouse Splenocyte Proliferation in the Presence ofRecombinant PD-L1

Recombinant mouse PD-L1 (rm-PDL-1, cat no: 1019-B7-100; R&D Systems)were used as the source of PD-L1.

Requirement:

Mouse splenocytes harvested from 6-8 weeks old C57 BL6 mice; RPMI 1640(GIBCO, Cat #11875); DMEM with high glucose (GIBCO, Cat # D6429); FetalBovine Serum [Hyclone, Cat # SH30071.03]; Penicillin (10000unit/ml)-Streptomycin(10,000 μg/ml) Liquid (GIBCO, Cat #15140-122); MEMSodium Pyruvate solution 100 mM (100×), Liquid (GIBCO, Cat #11360);Nonessential amino acid (GIBCO, Cat #11140); L-Glutamine (GIBCO, Cat#25030); Anti-CD3 antibody (eBiosciences-16-0032); Anti-CD28 antibody(eBiosciences-16-0281); ACK lysis buffer (1 mL) (GIBCO, Cat #-A10492);Histopaque (density-1.083 gm/mL) (SIGMA 10831); Trypan blue solution(SIGMA-T8154); 2 mL Norm Ject Luer Lock syringe—(Sigma 2014-12); 40 μmnylon cell strainer (BD FALCON 35230); Hemacytometer (Bright line-SIGMAZ359629); FACS Buffer (PBS/0.1% BSA): Phosphate Buffered Saline (PBS) pH7.2 (HiMedia TS1006) with 0.1% Bovine Serum Albumin (BSA) (SIGMA A7050)and sodium azide (SIGMA 08591); 5 mM stock solution of CFSE: CFSE stocksolution was prepared by diluting lyophilized CFSE with 180 μL ofDimethyl sulfoxide (DMSO C₂H₆SO, SIGMA-D-5879) and aliquoted in to tubesfor further use. Working concentrations were titrated from 10 μm to 1μm. (eBioscience-650850-85); 0.05% Trypsin and 0.02% EDTA (SIGMA59417C); 96-well format ELISA plates (Corning CLS3390); BD FACS caliber(E6016); Recombinant mouse B7-H1/PDL1 Fc Chimera, (rm-PD-L1 cat no:1019-B7-100).

Protocol

Splenocyte Preparation and Culturing:

Splenocytes harvested in a 50 mL falcon tube by mashing mouse spleen ina 40 μm cell strainer were further treated with 1 ml ACK lysis bufferfor 5 mins at room temperature. After washing with 9 mL of RPMI completemedia, cells were re-suspended in 3 ml of 1×PBS in a 15 mL tube. 3 mL ofHistopaque was added carefully to the bottom of the tube withoutdisturbing overlaying splenocyte suspension. After centrifuging at 800×gfor 20 min. at room temperature, the opaque layer of splenocytes wascollected carefully without disturbing/mixing the layers. Splenocyteswere washed twice with cold 1×PBS followed by total cell counting usingTrypan Blue exclusion method and used further for cell based assays.

Splenocytes were cultured in RPMI complete media (RPMI+10% fetal bovineserum+1 mM sodium pyruvate+10,000 units/mL penicillin and 10,000 μg/mLstreptomycin) and maintained in a CO₂ incubator with 5% CO₂ at 37° C.

CFSE Proliferation assay:

CFSE is a dye that passively diffuses into cells and binds tointracellular proteins. 1×10⁶ cells/ml of harvested splenocytes weretreated with 5 μm of CFSE in pre-warmed 1×PBS/0.1% BSA solution for 10mins at 37° C. Excess CFSE was quenched using 5 volumes of ice-coldculture media to the cells and incubated on ice for 5 min. CFSE labelledsplenocytes were further given three washes with ice cold complete RPMImedia. CFSE labelled 1×10⁵ splenocytes added to wells containing eitherMDA-MB231 cells (1×10⁵ cells cultured in high glucose DMEM medium) orrecombinant human PDL-1 (100 ng/mL) and test compounds. Splenocytes werestimulated with anti-mouse CD3 and anti-mouse CD28 antibody (1 μg/mLeach), and the culture was further incubated for 72 h at 37° C. with 5%CO₂. Cells were harvested and washed thrice with ice cold FACS bufferand % proliferation was analysed by flow cytometry with 488 nmexcitation and 521 nm emission filters.

Data Compilation, Processing and Inference:

Percent splenocyte proliferation was analysed using cell quest FACSprogram and percent rescue of splenocyte proliferation by compound wasestimated after deduction of % background proliferation value andnormalising to % stimulated splenocyte proliferation (positive control)as 100%.

Background proliferation: Splenocytes+anti-CD3/CD28+PD-L1

Stimulated splenocytes: Splenocytes+anti-CD3/CD28 stimulation

Compound proliferation: Splenocytes+anti-CD3/CD28+PD-L1+Compound

Compound effect is examined by adding required conc. of compound toanti-CD3/CD28 stimulated splenocytes in presence of ligand (PDL-1)

Table 5 shows the rescue of mouse splenocyte proliferation inhibited byrecombinant mouse PDL1 using CFSE based assay:

TABLE 5 Percent rescue of splenocyte Compound proliferation @ 100 nMcompound No. concentration 1 95 2 94 3 58 4 49 5 53 8 68 9 73 10 89 1291

What is claimed is:
 1. A compound of formula (I)

or a pharmaceutically acceptable salt or a stereoisomer thereof;wherein, R₁ is a side chain of amino acid Ala, Ser, Thr or Leu; R₂ is aside chain of amino acid Asp, Glu, Gln or Asn; [Aaa] is an amino acidresidue selected from Ser, Asp, Ala, Ile, Phe, Trp, Lys, Glu or Thr; R₃is hydrogen or alkyl; each of R₄ and R₄′ independently are hydrogen oralkyl; both R_(a) and R_(a)′ are hydrogen; or together are an oxo (═O)group; both R_(b) and R_(b)′ are hydrogen; or together are an oxo (═O)group; L is

X is CH₂, O or S; R₅ is hydrogen or alkyl; m is an integer from 1 to 3;and n is an integer from 2 to
 20. 2. The compound according to claim 1,wherein the compound of formula (I) is a compound of formula (IA):

or a pharmaceutically acceptable salt or a stereoisomer thereof;wherein, R₁ is a side chain of amino acid Ala, Ser, Thr or Leu; R₂ is aside chain of amino acid Asp, Glu, Gln or Asn; [Aaa] is an amino acidresidue selected from Ser, Asp, Ala, Ile, Phe, Trp, Lys, Glu or Thr; R₃is hydrogen or alkyl; each of R₄ and R₄′ independently are hydrogen oralkyl; both R_(a) and R_(a)′ are hydrogen; or together are an oxo (═O)group; and both R_(b) and R_(b)′ are hydrogen; or together are an oxo(═O) group.
 3. The compound according to claim 1, wherein the compoundof formula (I) is a compound of formula (IB):

or a pharmaceutically acceptable salt or a stereoisomer thereof;wherein, R₁ is a side chain of amino acid Ala, Ser, Thr or Leu; R₂ is aside chain of amino acid Asp, Glu, Gln or Asn; and [Aaa] is an aminoacid residue selected from Ser, Asp, Ala, Ile, Phe, Trp, Lys, Glu orThr.
 4. The compound according to claim 1, wherein the compound offormula (I) is a compound of formula (IC):

or a pharmaceutically acceptable salt or a stereoisomer thereof;wherein, R₁ is a side chain of amino acid Ala, Ser, Thr or Leu; R₂ is aside chain of amino acid Asp, Glu, Gln or Asn; and [Aaa] is an aminoacid residue selected from Ser, Asp, Ala, Ile, Phe, Trp, Lys, Glu orThr.
 5. The compound according to claim 1, wherein, R₁ is a side chainof amino acid Ser or Thr; R₂ is aside chain of amino acid Asp, Asn orGlu; [Aaa] is an amino acid residue Ser or Thr; R₃, R₄ and R₄′independently are hydrogen; both R_(a) and R_(a)′ together represent anoxo (═O) group; both R_(b) and R_(b)′ together represent an oxo (═O)group; L is —C(O)—(CH₂)_(m)—(X—CH₂—CH₂)_(n)—NH—; X is CH₂ or O; m is aninteger from 1 to 3; and n is an integer from 2 to 20; or apharmaceutically acceptable salt or a stereoisomer thereof.
 6. Thecompound according to claim 1, wherein R₄ is C₁₋₅ alkyl; or R₄′ is C₁₋₅alkyl.
 7. A pharmaceutical composition comprising at least one compoundaccording to claim 1 or a pharmaceutically acceptable salt or astereoisomer thereof, and a pharmaceutically acceptable carrier orexcipient.
 8. The pharmaceutical composition according to claim 7further comprising at least one of an anticancer agent, chemotherapyagent, or antiproliferative compound.
 9. A compound having a structuralformula selected from the group consisting of Com- pound No. Structure 1

 2

 3

 4

 5

 6

 7

 8

 9

10

11

12

13

14

15

16

17

18

19

20

21

22

23

24

25

or a pharmaceutically acceptable salt or a stereoisomer thereof.
 10. Apharmaceutical composition comprising at least one of the compoundsaccording to claim 9 and a pharmaceutically acceptable carrier orexcipient.